TNF-α is required for late ischemic preconditioning but not for remote preconditioning of trauma1

Journal of Surgical Research 121, 120 –129 (2004) doi:10.1016/j.jss.2004.03.010

TNF-␣ Is Required for Late Ischemic Preconditioning but Not for Remote Preconditioning of Trauma 1 Xiaoping Ren, M.D., Yang Wang, M.D., and W. Keith Jones, Ph.D. 2 Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, Ohio Submitted for publication February 2, 2004

Background. Ischemic preconditioning (IPC) and remote IPC are cardioprotective phenomena in which ischemia of the myocardium or of a remote tissue, respectively, induces cardioprotection. Despite clinical evidence that surgical trauma can remotely affect myocardial infarction, to date there are no basic science studies addressing the effect of nonischemic trauma at distant sites upon cardiac ischemia/ reperfusion (I/R) injury. The objectives of this study were to determine the effects of nonischemic remote surgical trauma upon infarct size after myocardial I/R and to determine the effects of TNF-␣ ablation upon cardioprotective phenomena. Materials and methods. A minimally traumatic mouse model was used to ascertain the effect of remote nonischemic surgical trauma upon I/R injury. TNF-␣ knockout mice were employed to determine the effect of TNF-␣ ablation. Results. Carotid artery vascular surgery remotely exacerbates cardiac I/R injury increasing infarct size by 287% (remote cardiac injury or RCI). Nonischemic, nonvascular trauma (abdominal incision) results in remote preconditioning of trauma (RPCT), decreasing infarct size by 81% (early phase) and 40% (late phase) relative to controls. Finally, TNF-␣ is required for late IPC but is not necessary for RCI or for RPCT. Conclusions. We show that late IPC is TNF-␣dependent and describe two unique TNF-␣-independent remote effects of nonischemic trauma upon myocardial infarction. Understanding the mechanism of these remote effects will allow the development of novel therapies for the treatment of ischemic heart disease. RPCT and TNF-␣ ablation have an additive protective effect 1 Funded by National Institutes of Health Grant HL63034-01 (W.K.J.). 2 To whom correspondence and reprint requests should be addressed at Department of Pharmacology and Cell Biophysics, 231 Albert Sabin Way ML0575, University of Cincinnati, Cincinnati, OH 45267-0575. E-mail: [email protected].

0022-4804/04 $30.00 © 2004 Elsevier Inc. All rights reserved.

suggesting that combinations of complementary approaches may be a useful strategy for maximizing the clinical efficacy of cardioprotective therapies. © 2004 Elsevier Inc. All rights reserved.

Key Words: surgical trauma; myocardial infarction; ischemia/reperfusion; ischemic preconditioning; remote preconditioning; TNF-alpha.

INTRODUCTION

Ischemic preconditioning (IPC) has been extensively studied since Murry et al. [1] first reported this phenomenon in 1986. IPC is a powerful cardioprotective phenomenon that has been successfully applied in multiple species [2– 6] and organs [7–9], and in the clinical setting [10 –12]. There are two phases of IPC: early IPC results in acute (1–2 h) protection against myocardial infarction (MI) [3, 5, 13], while late IPC results in long-lasting (2–3 days) protection against MI and stunning [2, 4, 5]. Recently, there has been recognition of a third type of cardioprotection, remote IPC that results from brief episodes of ischemia affected at a distant organ site, including kidney [14, 16 –18], intestine [19 – 21], or limb [15, 24]. Despite clinical data suggesting that nonischemic surgical trauma affects MI, this phenomenon has not been subjected to rigorous study in animal surgical models. Results of retrospective clinical studies suggest that surgical trauma induced at a remote site may have deleterious effects upon MI [23, 24]. In fact, patients recovering from surgery to repair abdominal aortic aneurysm have a significantly higher risk for MI, and on average have larger infarcts and increased mortality relative to those not having undergone such surgery [23–25]. We observed that infarct size after in vivo ischemia/reperfusion (I/R) was increased by remote nonischemic vascular surgery performed to catheterize

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the carotid artery for blood pressure measurements, a phenomenon we call remote cardiac I/R injury (RCI). RCI is distinguished from the previously described phenomenon of remote IPC by the fact that RCI is injurious rather than protective and involves no organ or tissue ischemia. To study the effect of surgical trauma upon development of MI, we developed a minimally traumatic mouse I/R model that has several advantages over previous models [5, 26], including reduced blood loss and lower mortality. Our studies demonstrated that a second nonischemic, nonvascular surgical trauma, transverse abdominal incision, applied either 15 min or 24 h before I/R, resulted in decreased infarct size. RPCT is different from the previously described phenomena of IPC and remote IPC because it does not involve organ or tissue ischemia to initiate protection. However, remote preconditioning of trauma (RPCT) is a preconditioning phenomenon just as IPC and remote IPC, as there is both an early and a late phase [14 –16, 20, 21, 27]. We designated these phenomena early and late RPCT. To our knowledge this is the first demonstration of remote preconditioning phenomena related to nonischemic trauma. The response to wounding involves the release of a variety of cytokines, chemokines, and endocrine factors from the wound site [28, 29]. TNF-␣ is a major cytokine that is synthesized and released by wounding [28 –30], is secreted into the bloodstream [30] involved in early IPC [31, 32], and mediates both protective and injurious effects in the heart [32–35]. We therefore tested the hypothesis that TNF-␣ release mediates these newly discovered remote effects. The results of these experiments demonstrate that TNF-␣ is not necessary for RCI or for RPCT. However, we demonstrate for the first time that TNF-␣ is necessary for late IPC. This suggests a novel cardioprotective mechanism for RPCT distinct from that of ischemic PC.

MATERIALS AND METHODS Animals Mice were maintained according to institutional guidelines, the Guide for the Care and Use of Laboratory Animals (NIH, 1985) and the Position of the American Heart Association on Research Animal Use (1984). TNF-␣ ⫺/⫺ mice, the generous gift of Dr. George Kolias (Hellenic Pasteur Institute), were maintained and genotyped as described [36]. Wild-type (WT) mice were obtained from Jackson Laboratories (B6129SF2/J F 2, strain 101045, Bar Harbor, ME) and are as close as possible to the genetic background of the TNF-␣ ⫺/⫺ mice. Males and females were distributed equally among groups and were between 10 and 16 weeks of age. Analysis by ANOVA showed no differences in infarct size in vivo between male and female mice in any experimental group, indicating, as previously reported [37], that there was no effect of sex upon infarct size in vivo. Based upon this, data from both sexes were pooled for each group.

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Mouse Model and Surgical Procedures Ischemic preconditioning and ischemia/reperfusion. Mice were anesthetized with sodium pentobarbital (90 mg/kg ip), intubated with PE 90 tubing, and ventilated using a mouse miniventilator (Harvard Apparatus, Holliston, MA). Blood pressure and ECG (DigiMed Sinus Rhythm Analyzer, Micro-Med, Inc., Louisville, KY) data were continuously recorded 10 min before and during ischemia and for 10 min after reperfusion. Although we found small decreases in blood pressure during ischemia, these were less than that reported previously (maximally 15 mmHg in this study versus 20 – 40 mmHg) [5], presumably due to the extremely small blood loss (estimated at 50 ␮l) from minimally invasive procedures. Though mean arterial blood pressure was significantly increased by saline iv, there was no effect of the 15 mmHg blood pressure drop upon infarct size after 30 min ischemia and 24 h reperfusion (45.1 ⫾ 3.4, with saline versus 40.7 ⫾ 4.3, without P ⫽ 0.18). The ventilation conditions used were as follows: rate ⫽ 100 ⫾ 5 breaths/min (closed system) [39], tidal volume ⫽ 2.2 ml [5, 26]. Blood gas analysis performed after openchest surgery and 30 min I/R using a 248 pH/Blood Gas Analyzer (Essex Co., 9 2DX, UK) gave values of pH ⫽ 7.4 ⫾ 0.01, P CO2 ⫽ 26.5 ⫾ 1.3, P O2 ⫽ 239.2 ⫾ 25.8 (n ⫽ 13). A lateral thoracotomy (1.5 cm incision between the second and third ribs) was performed to provide exposure of the left anterior descending coronary artery (LAD) while avoiding rib and sternal resection, retraction, and rotation of the heart. Vascular bundles in the vicinity were coagulated using a microcoagulator (Medical Industries, Inc., St. Petersburg, FL). An 8-O nylon suture was placed around the LAD 2–3 mm from the tip of the left auricle and a piece of soft silicon tubing (0.64 mm ID, 1.19 mm OD) was placed over the artery. Coronary occlusion was achieved by tightening and tying the suture. On day 1, mice in PC sham groups underwent thoracotomy and placement of the suture and the chest remained open for 44 min (no occlusion). Mice in PC groups underwent occluder placement followed by a series of six 4-min coronary occlusions interspersed with 4 min of reperfusion [1]. At the end of these procedures, the suture was left in place and the chest was closed in layers using 7-O polypropylene sutures. On day 2 mice were subjected to a second open-chest surgery involving a 30-min coronary occlusion. At the end of the occlusion, the suture was untied and left in place. Ischemia was confirmed by visual observation (i.e., by cyanosis) and by continuous ECG monitoring (widening of the QRS complex, T wave inversion, and ST segment changes) and reperfusion by reversal of these effects. Mice with unsuccessful coronary occlusion and/or reperfusion were excluded from the study (see below). The chest was closed in layers and the mice were allowed to regain consciousness in a warm chamber with 100% oxygen. Mice were euthanized after 24 h of reperfusion, stained, and sectioned for infarct size determination as described [5]. Sections were photographed using a Nikon Coolpix880 digital camera fitted with UR-E2 macro lens and computerized digital planimetry was performed using NIH Image software. Infarct size was determined by the method of Fishbein et al. and expressed as a percentage of the region at risk, since size of the risk region can be a variable for overall infarct size [40]. Carotid artery/jugular vein catheterization. A 1-cm transverse incision was made on the left side of the neck, and the jugular vein and carotid were exposed. The vessels were ligated superior to the point of catheter insertion and a piece of sterile PE 10 tubing was inserted into each vessel. The catheters were tied in place using an 8-O nylon suture. The tube inserted into the carotid was connected to a pressure transducer with a Digi-Med Blood Pressure Analyzer (Micro-Med). The tube inserted into the jugular was connected with a syringe containing saline. When a catheter was removed, the suture holding it in place was tightened to ligate the vessel. This surgery was performed with minimal blood loss, estimated at less than 50 ␮l.

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TABLE 1 Values for Infarct Size, Risk Region, and Normalized Infarct Size (Materials and Methods) for the Experimental Groups Used in This Study Group

Procedure Day 1

Infarct/LV

Risk/LV

Infarct/Risk

P versus control (Risk/LV)

P versus control (Infarct/Risk)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

PC Sham PC RCI Sham RCI Early RPCT Late RPCT Sham RPCT PC Sham PC RCI Sham RCI Early RPCT Late RPCT Sham RPCT

4.9 ⫾ 0.96 13.3 ⫾ 2.4 26.0 ⫾ 3.2 5.9 ⫾ 1.7 6.3 ⫾ 1.7 19.3 ⫾ 2.3 27.4 ⫾ 1.9 8.6 ⫾ 1.6 7.2 ⫾ 1.7 17.2 ⫾ 0.93 5.6 ⫾ 0.88 1 ⫾ 0.08 9.0 ⫾ 1.5 19.0 ⫾ 2.0

42.1 ⫾ 7.8 49.8 ⫾ 4.7 46.9 ⫾ 4.7 41.1 ⫾ 3.3 57.2 ⫾ 2.6 62.1 ⫾ 5.2 52.1 ⫾ 1.9 47.7 ⫾ 5.1 52.8 ⫾ 5.2 52.5 ⫾ 3.3 55.1 ⫾ 5.4 54.7 ⫾ 4.1 45.7 ⫾ 3.8 54.1 ⫾ 1.4

11.5 ⫾ 1.7 24.6 ⫾ 2.7 55.0 ⫾ 4.1 14.2 ⫾ 3.9 10.4 ⫾ 2.5 33.2 ⫾ 6.3 52.4 ⫾ 2.6 15.0 ⫾ 1.8 15.3 ⫾ 1.3 32.3 ⫾ 0.91 10.4 ⫾ 1.4 1.9 ⫾ 0.17 16.9 ⫾ 2.9 32.5 ⫾ 3.6

0.472 Control for 1 0.35 Control for 3 0.17 0.19 Control for 5,6 0.53 Control for 8 0.72 Control for 10 0.92 0.066 Control for 12,13

0.0027 Control for 1 ⱕ0.0001 Control for 3 ⱕ0.0001 0.0273 Control for 5,6 0.89 Control for 8 ⬍0.0001 Control for 10 ⱕ0.0001 0.0026 Control for 12,13

Note. Data presented as mean ⫾ SE. The final two columns present results of t-tests comparing the size of the risk region and the normalized infarct size between each experimental and the appropriate control group. Groups 1–7 are wild-type and groups 8 –14 TNF-␣ knockout mice. There were no significant differences in size of the risk region in experimental versus control groups. Abbreviations: Infarct/LV ⫽ mean area of the infarct divided by mean area of the left ventricle; Risk/LV ⫽ mean area of the risk region divided by mean area of the left ventricle; Infarct/Risk ⫽ mean area of the infarct divided by the mean area of the risk region; P versus control; the P value for the comparison between the indicated group and its control group (see Statistical Methods). This is shown for both the Risk/LV and for the Infarct/Risk calculations.

Abdominal incision. The abdominal incision used as the stimulus for RPCT was a 2-cm transverse incision located on the abdominal midline. The incision extended through the skin and muscle and into the peritoneum and was immediately closed using 7-O polypropylene sutures. Severed vessels were cauterized immediately and bleeding was minimal (⬍50 ␮l). The coronary occlusion time was increased from 30 to 45 min for RPCT studies because infarcts in TNF-␣ ⫺/⫺ mice after early RPCT were too small to measure (i.e., less than 1% of the risk region).

Experimental Design Effect of surgical procedures upon infarct size. WT mice were used in groups 1–7 and TNF ⫺/⫺ mice were used in groups 8 –14. Mice in groups 1 and 8 (n ⫽ 6) were subjected to preconditioning, while mice in groups 2 and 9 (n ⫽ 8 and n ⫽ 7) were subjected to sham surgery on day 1 (sham for IPC, see refers. [5 and 38]) Mice in groups 1, 2, 8, and 9 were subjected to a 30-min coronary occlusion on day 2. Mice in groups 3 and 10 (n ⫽ 6 and n ⫽ 8) were subjected to carotid catheterization followed by 30 min of coronary occlusion. Mice in groups 4 and 11 (n ⫽ 10 and n ⫽ 8) underwent 30 min of coronary occlusion without placement of catheters (sham for RCI). Mice in groups 5 and 12 (n ⫽ 6) underwent surgical abdominal incision followed 15 min later by 45 min of coronary occlusion. Mice in groups 6 and 13 (n ⫽ 6 and n ⫽ 12) underwent surgical abdominal incision on day 1 and were subjected to 45 min of coronary occlusion on day 2. In pilot studies, separate control groups for early RPCT (no abdominal incision, same anesthesia and timecourse as group 5) and late RPCT (anesthesia and intubation on day 1 with no abdominal incision, I/R on day 2 as for group 6) were performed and infarct size was not different between these groups (55.3 ⫾ 3.4 versus 49.5 ⫾ 4.0, P ⫽ 0.33); that is, there was no effect of anesthesia and intubation 24 h prior to challenge coronary occlusion. Thus, the pooled data (group 7, n ⫽ 12) were used as control for groups 5 and 6. This did not change the statistical significance of any of the comparisons per-

formed. Similarly, for TNF-␣ knockout mice (groups 12 and 13) mice in group 14 (n ⫽ 13) served as the control for both early and late RPCT. For all groups, infarct size was determined 24 h after reperfusion.

Statistical Methods Group sizes were determined by power analysis as described [5]. All results are reported as mean values ⫾ SE. Data were analyzed with a one-way repeated-measure ANOVA followed by unpaired Student’s t test. A P value ⱕ 0.05 was considered statistically significant.

RESULTS

Importantly, no significant differences in the size of the risk regions were discerned between experimental groups in this study and their control groups (Table 1). Late Phase of Ischemic Preconditioning (IPC)

Late PC reduced infarct size from 24.6 ⫾ 2.7 to 11.5 ⫾ 1.7% (Fig. 1, group 1 versus 2, P ⱕ 0.05). Though infarct size was smaller in TNF-␣ ⫺/⫺ relative to WT mice (group 2 versus 9), TNF-␣ ablation abrogated the infarct-sparing effect of late IPC (Fig. 1, 15.0 ⫾ 1.8%, group 8 versus 15.3 ⫾ 1.3%, group 9, P ⫽ 0.089). Effect of Surgery upon Infarct Size; Remote Cardiac I/R Injury

We found that infarct size was increased in group 3 (carotid catheterization, i.e., nonischemic vascular in-

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FIG. 1. Infarct size as a percent of the risk-region in wild-type (WT) and in TNF-␣ ⫺/⫺ mice subjected to sham surgery (groups 2, 9) or late PC (groups 1, 8). Error bars indicate the standard error of the mean (SE) and significant differences, determined as described (Materials and Methods) are indicated (*). Below, graphical representation of the experimental design for the studies, see Materials and Methods for details. O ⫽ coronary occlusion.

jury) relative to group 4 (Fig. 2, 55.0 ⫾ 4.1% versus 14.2 ⫾ 3.9%; P ⱕ 0.05), confirming our initial observation that mice subjected to carotid artery catheterization have larger infarcts relative to noncatheterized mice. Infarct size is similarly increased after carotid catheterization in TNF-␣ ⫺/⫺ mice (Fig. 2, 32.3 ⫾ 0.91%, group 10 versus 10.4 ⫾ 1.4%, group 11, P ⱕ 0.05). Remote Preconditioning of Trauma

Infarct size was significantly reduced in mice after abdominal incision (nonischemic, nonvascular injury)

15 min (group 5, early RPCT) and 24 h (group 6, late RPCT) before a challenge coronary occlusion (45 min), relative to mice not subjected to abdominal incision (Fig. 3, 10.4 ⫾ 2.5%, group 5 and 33.2 ⫾ 6.3%, group 6, versus 52.4 ⫾ 2.6%, group 7; P ⱕ 0.05). Mice in groups 12–14 were used to investigate the effect of TNF-␣ upon the early phase of RPCT. The results demonstrate that abdominal incision 15 min prior to a challenge ischemia (45 min) reduces infarct size significantly relative to mice not subjected to abdominal incision (Fig. 3, 1.9 ⫾ 0.17%, group 12 versus 32.5 ⫾ 3.6%, group 14, P ⱕ 0.05). Simi-

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FIG. 2. Infarct size as a percent of the risk-region in wild-type (WT) and TNF-␣ ⫺/⫺ mice subjected to carotid artery catheterization for investigation of remote cardiac injury (RCI). Error bars show standard error of the mean and significant differences determined as described (Materials and Methods) are indicated (*). Infarct size is generally reduced by TNF-␣ ablation (group 11 versus 4, P ⱕ 0.05) and RCI significantly increases infarct size by 74% (WT) and 68% (TNF-␣ ⫺/⫺), respectively (P ⱕ 0.05). Below, graphical representation of the experimental design for the studies, see Materials and Methods for details. O ⫽ coronary occlusion.

larly, abdominal incision performed 24 h prior to challenge ischemia (45 min) reduces infarct size (Fig. 3, 16.9 ⫾ 2.9%, group 13 versus 32.5 ⫾ 3.6%, group 14; P ⱕ 0.05) in TNF-␣ ⫺/⫺ mice. Exclusions

The entire study utilized 198 mice, 70 for pilot studies and 128 mice for studies of late IPC, RCI, and RPCT. Of the 121 mice, 121 survived to the intended endpoint (survival rate ⫽ 94.5%). Of the seven mice that died, two succumbed during or shortly after anesthesia induction, three had serious atrial arrhythmias or lethal ventricular arrhythmias, one expired due to improper ventilation,

and one died due to operative complication. These deaths were evenly distributed among groups, no group having more than one; therefore, there was no selection bias that might affect intergroup differences in infarct size. Of the 121 surviving mice, 7 were excluded either because there were no specific ECG changes indicative of ischemia or reperfusion (5 mice) or due to failure of the staining procedure (2 mice). DISCUSSION

The major results of this study are as follows: (1) TNF-␣ activity is necessary for the late phase of IPC

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FIG. 3. Panels a, b, and c show serial sections from a single representative mouse heart from groups 12, 13, and 14 (TNF-␣ ⫺/⫺; early RPCT, late RPCT, and sham), respectively. The red region (TCA-positive) is noninfarcted tissue (live at fixation); the white was infarcted tissue (TCA-negative, dead), and the blue tissue was nonischemic. Thus the risk region is the area of red plus white and the ratio of white to red plus white is the normalized (by risk region) infarct size. Panel d shows graphic representation of infarct size results expressed as a percent of the risk-region (groups 5–14). Error bars show standard error of the mean and significant differences as determined (Materials and Methods) are indicated (*). The cardioprotective effect of RPCT is unaffected by TNF-␣ ablation (group 12 and 13 versus 14). Below, graphical representation of the experimental design for the studies, see Materials and Methods for details. O ⫽ coronary occlusion.

against MI and (2) two different nonischemic remote traumas have distinct effects upon infarct development after coronary occlusion that are TNF-␣independent. While nonischemic carotid vascular surgery results in increased infarct size, abdominal incision (nonischemic, nonvascular injury) results in reduced infarct size. Furthermore, this remote preconditioning phenomenon (RPCT) has both early

and delayed components, much like IPC and remote IPC. Role of TNF-␣ in Late IPC

Although implicated in I/R injury and cardioprotection [31, 34, 41– 44], TNF-␣ has not been shown to be required for late IPC in the heart. Recently, experi-

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ments using TNF-␣ ⫺/⫺ mice established that TNF-␣ is necessary for early IPC [26]. We show that our model demonstrates a robust effect of late IPC against MI (Fig. 1, group 1 versus 2, 53.3% reduction in infarct size, P ⱕ 0.05). Although TNF-␣ ablation results in a significant 38% reduction of infarct size (Fig. 1, sham TNF-␣ ⫺/⫺, group 9 versus sham wild-type, group 2), late IPC is not detectable in TNF-␣ ⫺/⫺ mice (Fig. 1, group 8 versus 9). There are two interpretations of this result: first, that TNF-␣ is necessary for development of late IPC against MI, and second, that TNF-␣ ⫺/⫺ mice are already cardioprotected such that IPC does not further reduce infarct size. The second interpretation is based upon the assumption that IPC and cardioprotection due to TNF-␣ ablation are both all-or-none effects and are not additive. However, our results (Fig. 3) demonstrate that infarct size in TNF-␣ ⫺/⫺ mice can be further reduced by RPCT. Furthermore, Braun et al. have recently demonstrated that the cardioprotective effects of blockade and activation of PKC␦ and PKC␧, respectively, additively reduce infarct size [45]. Finally, we have shown that cardioprotection due to blockade of NF-␬B and TNF-␣ are additive (unpublished data). Thus, cardioprotective phenomena are generally not all-or-nothing phenomena and may additively affect cardioprotection. The lack of additivity between the cardioprotective effects of IPC and TNF-␣ ablation implies that these cardioprotective phenomena are different mechanistically. In light of these results, we favor the interpretation that TNF-␣ is necessary for late IPC. The cardioprotective effect of TNF-␣ ablation is likely due to the fact that TNF-␣ contributes to injury after I/R as well [31, 41, 42, 45]. TNF-␣ is known to be an upstream activator of NF-␬B, which plays a role in I/R and IPC [47, 48] and regulates iNOS gene expression [36]. We have previously shown that iNOS is required for development of late IPC [38] and it is possible that TNF-␣ may elicit NF-␬B-dependent iNOS expression during the development of late IPC. Whether these signaling pathways and effectors are protective or injurious after I/R may depend upon the specific insult, the levels and kinetics of their induction or factors as yet undetermined. Additional work, beyond the scope of the current study, is needed to address this possibility. Remote Cardiac I/R Injury

This investigation was triggered by our observation that the surgical trauma associated with carotid vascular surgery (nonischemic vascular injury) increases infarct size. Our studies confirmed the existence of RCI due to vascular catheterization (Fig. 2, group 3 versus group 4). Because the microsurgical techniques employed minimize blood loss (⬍50 ␮l), we believe this is not involved in development of RCI. Though it is doubtful that unilateral carotid catheterization causes brain

ischemia, we cannot rule this out as a trigger for RCI at this time. Though these experiments addressed only the early phase of RCI, other results suggest the existence of delayed RCI; we have long noted, in studies of late IPC, that infarct size is increased in mice subjected to sham surgery (thoracotomy) 24 h prior to challenge coronary occlusion, relative to mice that are surgically naı¨ve. We see the same effect in this study (e.g., 24.6 ⫾ 2.4%, group 2 versus 14.2 ⫾ 4.2%, group 4, P ⱕ 0.05). Remote Preconditioning of Trauma

To broaden the study and specifically to determine whether other types of surgical trauma elicit RCI, we investigated the effect of nonischemic nonvascular injury (abdominal incision) performed 15 min before a challenge coronary occlusion upon infarct size. The results demonstrate that abdominal incision elicits a powerful cardioprotective effect (Fig. 3, group 5 versus group 7), reducing infarct size by 81% (P ⱕ 0.05), an effect we designated early RPCT. Because of our interest and the great clinical relevance of late IPC, we tested the hypothesis that RPCT exhibits a delayed phase of protection against MI. Our results show that mice subjected to abdominal incision followed 24 h later by a 45-min challenge coronary occlusion have a 40% reduction in infarct size relative to controls (Fig. 3, group 6 versus group 7, P ⱕ 0.05). The decrease in infarct size due to late RPCT is in the vicinity of what is seen for late IPC [1, 4, 5, 20]. In both instances, the remote cardioprotection is truly a preconditioning phenomenon as an earlier treatment (nonischemic nonvascular abdominal incision) results in protection against a subsequent ischemic stress. RPCT is similar to recently described examples of remote IPC, in which ischemia induced at a remote site (e.g., intestine, limb, or kidney) results in protection against subsequent MI [14, 15, 17, 22], but RPCT does not involve an ischemic insult as an initiator. Wang et al. were the first to show a late remote PC effect in the heart, consequent to mesenteric artery occlusion [20]. This cardioprotective effect was somewhat weaker than that produced by the late RPCT effect (25% versus 40% reduction in infarct size) and was abrogated by iNOS inhibition [20], as we previously demonstrated for late IPC [36]. While this study was being performed, Kuntscher et al. reported that a 10-min hindlimb ischemic episode could induce late remote PC in rat cremaster muscle, indicative of a late remote PC phenomenon [27]. We have shown, for the first time, that a late remote PC effect occurs in the heart after specific nonischemic surgical trauma. The fact that cardioprotective or injurious effects of nonischemic surgical trauma have not been reported in previous studies [14 –22] likely reflects specific surgical procedures or the models employed. For instance, Patel et al. found no difference in infarct size between rats subjected to sham surgery for mesenteric artery occlu-

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sion and rats not subjected to surgery [21]. However, all rats in this study underwent arterial catheterization. It is possible therefore that effects of catheterization masked the effects on infarct size of other surgical procedures. We have noted in our studies of late IPC that, although vascular catheterization and sham thoracotomy increase infarct size, there is no effect of catheterization in mice subjected to prior sham thoracotomy, suggesting that one effect masks the other. Interactions between cardioprotective and injurious signaling elicited by various types of surgical trauma may underlie clinical observations that results of ischemic preconditioning are not predictable in cardiac surgery [22]. A complete understanding of how specific surgical traumas and combinations of traumas affect MI will require further study. Role of TNF-␣ in RPCT

Whether TNF-␣ is primarily injurious or protective in the setting of cardiac I/R remains controversial and it is likely that the effect of TNF-␣ depends upon the status of other signaling pathways and environmental factors [31, 41, 42, reviewed in 43]. TNF-␣ is synthesized and released from many cell types, including muscle, cardiomyocytes, endothelial cells, and inflammatory and infiltrating cells [28, 33, 49]. This suggested the possibility that TNF-␣ may mediate both the remote injurious and the cardioprotective phenomena described herein (i.e., RCI and RPCT). However, experiments with TNF-␣ ⫺/⫺ mice showed that, although there was a general reduction in infarct size, suggestive of an overall ameliorative effect of TNF-␣ abrogation upon I/R injury (e.g., group 9 versus 2, Fig. 1 and group 14 versus 7, Fig. 3), TNF-␣ is not necessary for either RCI or RPCT (Figs. 2 and 3). Interestingly, early RPCT in TNF-␣ ⫺/⫺ mice reduced infarct size from 32.5 ⫾ 3.6% (group 14) to 1.9 ⫾ 0.17% of the region at risk (Fig. 3, group 12, P ⱕ 0.05). This amounts to a 94% reduction in infarct size and is, to our knowledge, the most powerful cardioprotective effect yet documented. Similarly, the cardioprotective effects of late RPCT (group 6 versus 7, 40% reduction) and TNF-␣ ablation (group 9 versus 2, 37.8% reduction) are independent and reduce MI in a nearly additive fashion (group 13 versus 7 is a 69.4% reduction). Thus, RPCT is TNF-␣independent and the additivity of the cardioprotective effects of TNF-␣ ablation and RPCT underscores that these phenomena are mechanistically different. RPCT and the Basis of Remote Effects

The results of these studies suggest that nonischemic nonvascular surgical trauma produces one or more factors that ameliorate cardiac I/R injury at a distance. At present we do not know the identity of these factors, nor whether the same factors are involved in RCI, early and late RPCT. Schoemaker and

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van Heijningen demonstrated that bradykinin is necessary for development of early remote IPC in the heart after mesenteric artery occlusion [18]. The fact that bradykinin administered via the mesenteric artery mimics remote PC, and that this effect can be blocked by the ganglion blocker hexamethonium, supports the authors’ hypothesis that bradykinin is released into the mesenteric vascular bed during mesenteric I/R, stimulating efferent nerves to the heart. Though not yet implicated in late IPC, norepinephrine has been shown to be involved in the mechanism of early IPC [50] and PC in the heart can be mimicked by catecholamine release [51]. Therefore, it is possible that catecholamines and sympathetic neuronal activity may be involved in RPCT. This idea is intriguing as norepinephrine has been implicated in signaling crosstalk with PKC [50], and nitric oxide (NO) has been shown to modulate norepinephrine release in skeletal muscle during ischemia [52]. Since both PKC and NO are known to be critically involved in late IPC [5, 38, 53], it is interesting to speculate that these molecules mediate aspects of RPCT. Another potential mediator of RPCT is adenosine [17], which has been shown to be TNF-␣-independent [32] and in fact represses TNF-␣ release from macrophages and production in the heart postischemia [54, 55]. Recent investigations have implicated adenosine receptors in early remote PC in the heart due to renal ischemia [17] and opioids in remote PC due to mesenteric artery occlusion [21]. Importantly, the fact that TNF-␣ ablation has no effect upon development of RCI and RPCT indicates that these phenomena are mechanistically different from IPC, since both early and late IPC require TNF-␣ [32 and this study]. Future studies will address the mechanism of the remote effects of surgery upon MI. Clinical Implications

Our results with RCI indicate that specific remote nonischemic vascular surgical trauma can exacerbate I/R injury in the heart. This suggests a rational basis for clinical observations that patients having undergone surgery for abdominal aortic aneurysm, for instance, are at increased risk for MI and have large infarcts [24, 25]. Although the abdominal incision and infrarenal ischemia/reperfusion involved in this surgery would be expected to be cardioprotective [14, 16 – 18], our data suggest that the effect of the vascular injury (to the aorta) overrides the protective effect of I/R. Further study is needed to determine the mechanism of these injurious effects and whether they can be ameliorated by cardioprotection and/or preconditioning. On the other hand, our results with RPCT demonstrate a novel and heretofore unsuspected preconditioning effect. It seems, generally speaking, that many types of stress elicit cardioprotection. In addition to IPC and remote IPC, it has been shown that hemor-

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rhage induces cardioprotection against ventricular dysfunction post-I/R via alpha 1-adrenergic signaling [56]. Whether the mechanisms that underlie RPCT, which does not involve significant hemorrhage, and the cardioprotection that occurs subsequent to hemorrhage are related mechanistically is unknown. Particularly, the results of applying early RPCT to TNF-␣ ⫺/⫺ mice are astonishing; a nearly complete cardioprotection against MI effected by a 45-min coronary occlusion! This highly efficacious cardioprotection is due to the simultaneous induction of RPCT and the removal of the injurious effects of TNF-␣. This also accounts for the additivity of the protective effects against MI of late RPCT and TNF-␣ ⫺/⫺ gene ablation. These results suggest that clinical efficacy of cardioprotective treatments may be maximized by using complementary approaches with additive effects. Although a measure of cardioprotection has been shown to result from anesthesia, cardioplegia, and cardiopulmonary bypass [22], development of new pharmacological agents that work by novel mechanisms and take advantage of additive effects hold the promise to further reduce myocardial damage. Understanding the detailed mechanism of these remote effects is necessary to develop such approaches.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Summary

We show for the first time that late IPC is abrogated by genetic ablation of TNF-␣. Furthermore, we describe and investigate nonischemic remote phenomena that affect I/R injury in the heart. Our results demonstrate that TNF-␣ is not involved in mediating RCI or RPCT. The additivity of the cardioprotective effects against MI of TNF-␣ ablation and RPCT indicates that these phenomena are mechanistically different. Finally, combinations of agents that produce additive cardioprotection are likely to be useful in maximizing the clinical efficacy of cardioprotective treatments.

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